2004_Sartoratto_COMPOSITION AND ANTIMICROBIAL ACTIVITY

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Brazilian Journal of Microbiology (2004) 35:275-280
ISSN 1517-8382
275
COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS FROM AROMATIC
PLANTS USED IN BRAZIL
Adilson Sartoratto; Ana Lúcia M. Machado; Camila Delarmelina; Glyn Mara Figueira; Marta Cristina T. Duarte*;
Vera Lúcia G. Rehder
Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas – Universidade Estadual de Campinas, Campinas,
SP, Brasil
Submitted: April 01, 2003; Returned to authors for corrections: October 31, 2003; Approved: October 04, 2004
ABSTRACT
Essential oils from aerial parts of Mentha piperita, M. spicata, Thymus vulgaris, Origanum vulgare, O.
applii, Aloysia triphylla, Ocimum gratissimum, O. basilicum were obtained by steam destillation using a
Clevenger-type system. These oils were screened for antibacterial and anti-Candida albicans activity using
bioautographic method. Subsequently, minimal inhibitory concentration from oils was determined by
microdilution method. Most essential oil studied were effective against Enterococcus faecium and Salmonella
cholerasuis. Aloysia triphylla and O. basilicum presented moderate inhibition against Staphylococcus
aureus while only A. tryphila and M. piperita were able to control the yeast Candida albicans. The oils were
analyzed by GC and GC-MS techniques in order to determine the majoritary compounds.
Key words: essential oil, medicinal plants, antimicrobial activity, minimal inhibitory concentration
INTRODUCTION
Higher and aromatics plants have traditionally been used in
folk medicine as well as to extend the shelf life of foods, showing
inhibition against bacteria, fungi and yeasts (17). Most of their
properties are due to essential oils produced by their secondary
metabolism (1). Essential oils and extracts from several plant
species are able to control microorganisms related to skin (1),
dental caries (7), and food spoilage, including Gram-negative
and Gram-positive bacteria (14).
Many countries have maintained research programs to screen
traditional medicines for antimicrobial activity, as is the case of
India (2), Palestin (4), Africa (6), Honduras (19), Jordan (20),
Cuba (21) and Italy (23). Plants from Brazilian biomes have also
been used as natural medicines by local populations in the
treatment of several tropical diseases, including schistosomiasis,
leishmaniasis, malaria and fungal and bacterial infections (5).
However, despite the rich flora, only data from a few plants is
available, including both native and exotic species.
Medicinal plants from CPQBA/UNICAMP germoplasm
collection have been studied against bacteria and the yeast
Candida albicans (Robin) Berkhout ATCC 10231. Extracts,
fractions and compounds isolated from Mikania laevigata Sch.
Bip ex Baker, M. glomerata Sprengel, Artemisia annua L.,
Phyllanthus niruri L., Phyllanthus amarus Schumach. & Thonn
and Achyrocline satureioides (Lam.) DC were able to control
one or more microorganisms (11,12,13,24).
Aromatic plants and spices have great importance for food,
cosmetics and pharmaceutical industries. Their use have taken
place since ancient times, and despite many of them were
substituted by synthetic ones, the demand for natural products
is increasing (15). Leafs from O. vulgare L., O. applii L., O.
basilicum L., O. gratissimum L., M. spicata L. e M. piperita L.
var. citrata have been used as spices and teas after drying, while
the essential oil is utilized in cosmetics and pharmaceuticals. The
essential oils contents in different species is influenced by
genetic material, culture conditions and environment, (8) and
finally, by crop and post-crop processing (22).
*Corresponding author. Mailing address: Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas - CPQBA/UNICAMP. Caixa Postal
6171, 13.083-970, Campinas, SP, Brasil. Fax: (+5519) 3884-7811. E-mail: mduarte@cpqba.unicamp.br
276
A. Sartoratto et al.
In the present study, the in vitro antimicrobial activity of
the essential oils from eight aromatic plants used in Brazil was
investigated. The levels and oil composition were characterized
by gas-chromatography/mass spectrophotometrical analyses.
MATERIALS AND METHODS
Aromatic Plants
Mentha piperita L. (UEC 127.110), M. spicata L. (UEC
121.401), Thymus vulgaris L. (UEC 121.405), Origanum vulgare
L. (UEC 121.409), O. applii (Domin) Borus (UEC 121.410),
Aloysia triphylla (L´Hér.) Britton (UEC 121.412), Ocimum
gratissimum L. (UEC 121.407) and O. basilicum L. (UEC
121.408) were chosen to the present study. The aromatic plants
were collected from CPQBA/UNICAMP experimental field,
between 9:00 and 10:00 am, in the first week on March, in full
flowering, except to O. vulgare L. in vegetative stage.
Essential oil extraction
The oil extraction was obtained from 40g fresh plants by steam
destillation using Clevenger system, during 3 h. The aqueous
phase was extracted with dichloromethane (3 x 50 mL). The organic
phase was dried with sodium sulphate, filtered and the solvent
evaporated until dryness. The oil was solubilized in ethyl acetate
for gas chromatography and mass spectrometry analysis.
Chromatography conditions
The identification of volatile constituents was conducted by
gas-chromatography in Hewlett-Packard 5890 Series II (Palo Alto,
CA, USA) equipment, with selective mass detector HP-5971 in
the electron impact (EI) ionization mode (70 eV), injector split/
splitless, capillary column HP-5 (25 m x 0.2 mm x 0.33 µm).
Temperature: injector = 220ºC, column = 60ºC, 3ºC.min-1, 240ºC
(7 min). Carrier gas (He) = 1.0 mL.min-1. Retention indices (RI) have
been obtained according to the method of Van den Dool (26).
Microorganisms
Antimicrobial activity tests were carried out against the
bacteria Pseudomonas aeruginosa (Schroeter) ATCC13388,
Salmonella choleraesuis (Smith) CCT4296, Rhodococcus equi
(Magnussom) CCT0541, Micrococcus luteus (Schroeter)
CCT2692, Staphylococcus aureus (Rosenbach) CCT2740, S.
epidermides (Winslow & Winslow) ATCC12228, Escherichia
coli CCT0547, Bacillus subtilis (Ehrenberg) Cohn CCT2576,
Enterococcus faecium ATCC10541 (Orla-Jensen) Schleifer and
Kilpper-Balz (registered at ATCC as Streptococcus faecium),
Enterococcus faecium (Orla Jensen) CCT5079 and against the
yeast Candida albicans (Robin) Berkhout ATCC 10231.
Culture Media
Bacteria were assayed on Nutrient Agar (Merck, g/L):
peptone from meat, 5.0; meat extract, 3.0 and agar-agar, 12.0,
and C. albicans on Sabouraud Dextrose Agar (Merck, g/L):
peptone, 10.0; glucose, 40.0; agar-agar, 15.0.
Inocula
Inocula for the assays were prepared by diluting scraped
cell mass in 0.85% NaCl solution, adjusted to McFarland scale
0.5 and confirmed by spectrophotometrical reading at 580 nm.
Cell suspensions were finally diluted to 104 UFC.mL-1 for being
used in the activity assays.
Bioautography assays
Antibacterial activity tests were carried out prior by
bioautography method on thin layer chromatography (TLC)
plates (25). After dilution in ethyl acetate, the essential oils (3
µL at 10 mg/mL) were applied on duplicate TLC plates, and
hexane:acetate (85:15, v/v) was used as eluent. Subsequently,
the first plate was developed with anisaldehyde and the other
one was submitted to microbiological assays. Chloramphenicol
was used as positive control.
The bacterial inocula suspensions prepared as described
were inoculated by pour-plate in the respective medium (1:100)
around 42ºC. A 0.5 mL aliquot of 1 mg/mL trifenil tetrazolium
chloride solution (TTC) was added as growth indicator. The
media were transferred to the Petri dishes where the TLC plates
were previously deposited. After homogenization, the cultures
were incubated at 37ºC during 24h.
The oils activity against C. albicans (Robin) Berkhout was
evaluated only by MIC (Minimal Inhibition Concentration) test.
Minimal Inhibitory Concentration (MIC) Tests
MIC tests were carried out according to Eloff (10), using a
tissue culture testplate (96 wells). The stock solutions of the
oils were diluted and transferred into the first well, and serialdilutions were performed so that concentrations in the range of
2-0.03 mg.mL-1 were obtained. Chloramphenicol or nistatin
(Merck) was used as the reference antibiotic control. The
inoculum was added to all wells and the plates were incubated
at 37ºC during 24 h (bacteria) or at 30ºC for 48 h (yeast).
Antimicrobial activity was detected by adding 20 µL of 0.5%
TTC (triphenyl tetrazolium chloride, Merck) aqueous solution.
MIC was defined as the lowest concentration of oil that inhibited
visible growth, as indicated by the TCC staining.
RESULTS AND DISCUSSION
Oil Yield and Chemical Constituents
Oil yields expressed in relation to dry weight plant material
are shown in Table 1. The highest (0.74% w/w) and lowest
(0.10% w/w) yields were obtained from O. gratissimum L. and
O. basilicum L., respectively.
The chemical composition of the essential oils obtained
was analyzed by GC and GC-MS, which allowed identification
Antimicrobial activity of aromatic plants
277
of about 80% of oil constituents (Table 2). The main
compounds from Aloysia triphylla (L´Hér.) Britton oil were
geranial (21.83%), neral (17.45%) and limonene (11.03%).
Thymus vulgaris L. main components were thymol (79.15%),
carvacrol (4.63%) and p-cimene (3.27%). The Mentha species
showed different oil composition. Linalool (51.0%), carvone
(23.42%) and 3-octanol (10.1%) were identified from M. piperita
L. and piperitenone oxide (94.8%) was present in M. spicata L.
Thymol was the main constituent of O. applii (Domin) Borus
(64.5%) and O. vulgare L. (38.0%) while eugenol was the
component obtained from O. gratissimum (93.9%) and O.
basilicum L. (28.1%).
Antimicrobial activity
Many microorganisms, which cause damage to human
health, exhibit drug resistance due to inadequate use of
antibiotics. Thus, there is a need for the discovery of new
substances from natural sources, including plants. In this work,
the antimicrobial activity of essential oils from aromatic species
used in Brazil was previously evaluated by bioautographic assay
that allows identification of oils active fractions. The trifenil
tetrazolium chloride indicates cellular growth, once alive cells
turn red. Thus, white spots indicate regions where the oil fraction
was active. According to results the oils presented one or more
active fractions against the microorganisms studied (Table 3).
Table 1. Essential oil concentration (% w/w) from aromatic plants studied, including the botanical name, family and traditional use.
Botanical name Family Traditional Use Essential oil (% w/w)
Aloysia tryphila (L´Hér.) Britton Verbenaceae spice, digestive, sedative 0.22
Thymus vulgaris L. Lamiaceae antiseptic, antispasmodic 0.56
Mentha piperita L. Lamiaceae antiseptic, vermifug 0.42
M. spicata L. Lamiaceae antispasmodic, diuretic 0.32
Ocimum basilicum L. Lamiaceae digestive, vermifug 0.10
O. gratissimum L. Lamiaceae anticold, diuretic 0.74
Origanum vulgare L. Lamiaceae analgesic, expectorant 0.13
O. applii L. Lamiaceae analgesic, expectorant 0.20
Table 2. Identified compounds from aromatic plants: AT = A. triphyla; TV = T. vulgaris; MS = M. spicata; MP = M. piperita; OB =
O. basilicum; OG = O. gratissimum; OV = O. vulgare and OA = O. applii. (a) RI = retention index; (b) Results expressed as % Area.
(b) Sample
Analyte (a) RI
AT TV MP MS OV OA OB OG
1-octen-3-ol 978 0.38 0.78 0.68
3-octanol 998 10.1
p-cimene 1024 1.13 3.27 1.50
limonene 1028 11.03
1,8-cineol 1031 1.05
trans-β-ocimene 1035 0.41 0.39
γ-terpinene 1057 0.47 2.57 1.99
isopentyl n-butirate 1068 0.67
α-terpinolene 1083 0.31
fenchone 1088 0.53
linalool 1093 1.05 1.62 51.0 4.92 32.6
cis-p-menth-2-en-1-ol 1122 0.52 1.47
trans-p-menth-2-en-1-ol 1140 0.86
camphor 1150 10.1
borneol 1169 2.29 2.52
Terpin-4-ol 1176 1.20 8.00 33.3 3.08 0.99 0.26
cimen-8-ol 1182 0.64
α-terpineol 1188 0.45 1.31 4.25 3.90
278
A. Sartoratto et al.
The MIC was determined only for oils that presented
positive results on bioautographic assays. Comparing with
literature results (3) strong activity is for MIC values between
0.05 – 0.50 mg/mL, moderate activity MIC values between 0.6 –
1.50 mg/mL and weak activity above 1.50 mg/mL.
The results show a variable effect of the oils on the
microorganisms (Table 3). Essential oil from Aloysia tryphila
(L´Hér.) Britton was active against six of the microorganisms
tested, showing the lowest MIC values (0.05 mg/mL and 0.50
mg/mL) against E. faecium ATCC 10541 (Orla-Jensen) Schleifer
& Kilpper-Balz and B. subtilis (Ehrenberg) Cohn, respectively.
T. vulgaris L., M. piperita L., O. gratissimum L., O. vulgare L. e
O. applii L. showed strong activity against E. faecium ATCC
10541 (Orla-Jensen) Schleifer & Kilpper-Balz (0.05 – 0.40 mg/
mL) and moderate activity against S. choleraesuis (Smith) (0.60
mg/mL) and S. aureus (Rosenbach) (1.00 mg/mL). All oils studied
presented moderate activity against S. aureus (Rosenbach) and
B. subtilis (Ehrenberg) Cohn, except M. spicata L. which was
inactive. The fact of M. spicata presents piperitone oxide (94.8%)
as main component and not shows any activity allows
concluding that it is not a antimicrobial compound. Finally,
Aloysia triphylla (L´Hér.) Britton and M. piperita L. exhibited
moderate activity against C. albicans (Robin) Berkhout (0.80
and 0.74 mg/mL, respectively).
Among the aromatic plants studied, the major constituents
found were the monoterpenes linalool, eugenol and thymol.
trans-piperitol 1208 0.16
thymol methyl ether 1231 0.76 0.24 1.47
neral 1238 17.45
carvacrol methyl ether 1241 1.33 5.91
carvone 1241 23.42
geranial 1270 21.83
thymol 1294 79.15 38.0 64.5
carvacrol 1298 4.63
eugenol 1358 28.1 93.9
piperitenone oxide 1373 94.8
Geranyl acetate 1379 3.29 0.33
β-bourbonene 1385 0.18 1.35 0.23
β-elemene 1393 1.92
Cedrene <1,7-di-epi-alpha> 1397 0.28
trans-caryophillene 1422 6.65 2.62 2.31 2.66 2.00 1.08
β-gurjunene 1430 0.36
β-bergamotene 1435 1.66
α-guaiene 1438 0.29
β-farnesene 1454 0.76
α-humulene 1455 0.77
allo-aromadendrene 1461 0.28
Germacrene D 1482 0.44 1.47 4.79 5.49 4.23
Curcumene <AR> 1483 5.14
γ-muurolene 1484 1.06
zingiberene 1495 0.51
δ-guaiene 1506 0.68
β-bisabolene 1508 1.05 1.98
γ-cadinene 1516 0.38 0.89 1.27
δ-cadinene 1524 0.57 0.29 0.19
espatulenol 1580 4.31 1.44 1.62
caryophillene oxide 1586 6.96 1.07
epi-α-muurolol 1643 0.72 5.81
α-eudesmol 1655 0.24
α-cadinol 1656 0.29 1.53 0.38 0.16
SUM OF IDENTIFIED PEAKS 81.9 94.6 98.0 96.6 95.3 94.1 98.3 99.9
Antimicrobial activity of aromatic plants
279
These compounds are previously known for its antimicrobial
activity (9,16,18). Helander et al. (16) attributed the thymol
antimicrobial action to its phenolic character, which can cause
membrane-disturbing activities.
Finally, regarding to effects of the antibiotics used as
positive control, A. tryphilla (L´Hér.) Britton presented MIC
value lower than chloramphenicol, showing the antimicrobial
potential of the essential oil.
Subsequently, bioguided fractionation will be conducted to
the potential plants for identification of the active compounds.
Evaluations of the oils from other medicinal plants are also being
conducted.
ACKNOWLEDGEMENTS
The authors would like to thank FAPESP, SP - Brazil for
financial support.
RESUMO
Composição e atividade antimicrobiana de óleos
essenciais de plantas aromáticas usadas no Brasil
Óleos essenciais foram obtidos a partir das partes aéreas de
Mentha piperita, M. spicata, Thymus vulgaris, Origanum
vulgare, O. applii, Aloysia triphylla, Ocimum gratissimum e
O. basilicum através de arraste de vapor em sistema tipo
Clevenger. Os óleos foram avaliados quanto à atividade
antimicrobiana contra bactérias e contra a levedura Candida
albicans pelo método de bioautografia. A concentração mínima
inibitória dos óleos com atividade positiva foi em seguida
determinada pelo método da microdiluição. De acordo com os
resultados, a maioria dos óleos essenciais estudados foram
Table 3. Bioautography and Minimal Inhibitory Concentration (MIC – mg.mL-1) of the essential oils from aromatic plants.
ControlsA. tryphila T. vulgaris M. spicata M. piperita O. basilicum O. gratissimum O. vulgare O. applii
Microorganism (Antibiotic MIC) B MIC B MIC B MIC B MIC B MIC B MIC B MIC B MIC
mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL
S. aureus 0.02 ++ 0.80 + 1.00 - > 2.00 ++ 1.00 + 0.70 + 1.00 ++ 1.00 ++ 1.00
E. faecium1 0.12 +++ 0.05 ++ 0.15 + > 2.00 +++ 0.15 + 1.00 ++ 0.30 ++ 0.40 ++ 0.20
S. choleraesuis 0.06 +++ 0.60 + 0.60 + > 2.00 ++ 0.60 + > 2.00 + 0.60 ++ 0.60 ++ 0.60
P. aeruginosa 0.85 - - - - - - - - - - - - - - - -
B. subtilis 0.02 + 0.50 + 0.49 + > 2.00 ++ 1.00 + 0.80 + 1.10 ++ 1.00 ++ 1.20
E. faecium2 0.07 ++ > 2.00 + > 2.00 - - - - - - - - - - - -
S. epidermidis 0.04 - - - - - - - - - - - - - - - -
R. equi 0.04 ++ > 2.00 + > 2.00 - - - - + > 2.00 +++ > 2.00 - - - -
M. luteus 0.05 ++ 1.60 + 2.00 - - + > 2.00 + 2.00 + > 2.00 + > 2.00 + > 2.00
E. coli 0.04 - - - - - - - - + > 2.00 ++ > 2.00 - - - -
C. albicans 0.05 - 0.80 - 2.00 - > 2.00 - 0.74 - > 2.00 - > 2.00 - 2.00 - > 2.00
+ one active fraction; ++ two active fractions; +++ three active fractions on Bioautography Test (B);
1E. faecium ATCC 10541; 2E. faecium CCT 5079.
efetivos contra Enterococcus faecium e Salmonella
cholerasuis. A.triphylla e O. basilicum apresentaram inibição
moderada contra Staphylococcus aureus enquanto apenas A.
tryphila e M. piperita foram capazes de inibir a levedura
Candida albicans. Os óleos foram analisados quimicamente
por técnicas de CG e CG-EM de modo a determinar os compostos
majoritários presentes.
Palavras-chave: óleos essenciais, plantas medicinais, atividade
antimicrobiana, concentração mínima inibitória
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